Liposome-enhanced immunoassay and test device

ABSTRACT

A test device for detecting or determining an analyte in a test solution includes an absorbent material having separate contact, competitive binding, and measurement portions. The contact portion is positioned for contact with and uptake of the test solution. The competitive binding portion has a binding material for the analyte non-diffusively bound thereto. The measurement portion has a receptor for the analyte and marker-encapsulating liposomes non-diffusively bound thereto. In a method for using the test device, a solution containing the analyte and the analyte-liposome conjugate is allowed to traverse the absorbent material from the contact portion through the competitive binding portion and on through the measurement portion of the absorbent material. The amount of marker in the measurement portion of the absorbent material, following traversal by the test solution, is then determined as a measure of the analyte in the sample. 
     Liposomes encapsulating an electroactive marker are used in conjunction with a test device as described above but which includes an electrochemical measurement portion in place of the measurement portion described above. Test devices and methods employing electrochemical detection or quantification of an electroactive marker corresponding to the amount of analyte in a sample may be either amperometric or potentiometric.

This invention was made with Government support under Grant No.1-P42-ES-05950-01, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a method for detecting or determiningone or more analytes, and a test device used in the method. Moreparticularly, the invention relates to a single-use test strip for usein an immunomigration assay employing marker-loaded liposomes for signalamplification.

BACKGROUND OF THE INVENTION

There is an increasing need for rapid, reliable, and inexpensive methodsfor detecting and measuring pollutants and contaminants in theenvironment and in food sources. Conventional analytical methods such ashigh pressure liquid chromatography, gas chromatography/massspectroscopy, atomic absorption spectroscopy, etc. are particularlyunsuitable for use in the field, because such methods are generallycomplex and employ instruments and equipment which are expensive andsusceptible to damage from transport and possible contamination in thefield. Gathering samples in the field for analysis at a remotelaboratory is similarly unsatisfactory, because it may take a few daysto several weeks from sample acquisition to obtain the results.

The need for simple, rapid, and inexpensive field assays has led to aninvestigation of immunoassays for surveying environmental contamination.Immunoassays comprise one category of specific binding assays, whichgenerally rely on the affinity of naturally occurring receptors orantibodies for specific compounds. The specific binding pairs employedin immunoassays are either an antigen or a hapten, and the antibodyproduced in immune response to the antigen or hapten.

Competitive immunoassays are generally based upon the competitionbetween a specific analyte, the amount of which is to be determined, anda labelled form of the analyte or an appropriate analog thereof, whichis used as an indicator, for a limited number of available binding siteson a binding material specific for the analyte. Using a known amount ofthe labelled analyte, the amount of analyte in the sample can bedetermined by measuring the amount of the unbound labelled analyte,which in some systems is physically separated from the bound indicatorduring the assay. Alternatively, where it is possible to distinguishbound from unbound indicator, such as where detectable physical orchemical changes in the indicator occur as a result of the bindingreaction, an assay can be completed without separating the bound andunbound indicator.

The types of materials commonly used as immunoassay label materials ormarkers include various enzymes, fluorescent dyes, chemiluminescentreactants, and radioisotopes. Such materials are often conjugated to theanalyte, as in the case of enzymes and radioisotopes, or lessfrequently, carried within sacs such as animal erythrocytes, polymermicrocapsules, or liposomes.

Immunoassays have been widely used for medical diagnosis for many years.More recently, immunoassays have been more broadly applied for thedetermination of toxic substances in the environment and in food.Practical applications for immunoassays in environmental analysisinclude evaluating the geographical scope and magnitude of pollutants,monitoring the fate and persistence of contaminants, and assessing theeffectiveness of remediation efforts. Raw and processed foods mustsimilarly be tested for chemical and biological contamination.

A wide variety of immunoassays, reagents, and test devices which exploitthe interaction between the members of specific binding pairs to detector measure a substance in a test sample have been developed.

Sophisticated, automated immunoassay systems are successfully employedin laboratory settings, but there are also many types of portablesensing devices which can be used outside the laboratory. Some portableimmunoassays and test devices have even been developed for use in thehome by untrained personnel. Home pregnancy test kits are an example ofsuch immunoassay test kits.

An immunochromatographic assay method for whole blood samples isdescribed in U.S. Pat. No. 4,594,327 to Zuk. At least one member of thespecific binding pair is uniformly bound to the entire surface of asolid bibulous element. The element is contacted with the whole bloodsample containing the analyte in an aqueous medium so that the sampletraverses the element to define a border related to the amount ofanalyte. The analyte concentration is directly related to the distancethe analyte has traversed. Zuk further describes determination of theborder by a separate development step, such as an enzyme or chromophoresignal production and amplification system.

U.S. Pat. No. 5,085,987 to Olson also describes an immunoassay employinga bibulous element such as a piece of paper affixed to plastic withadhesive. The element is contacted with the test solution suspected ofcontaining the analyte, to which has been added an antibody for theanalyte and a conjugate of the analyte and a label. The element containsa first receptor for the conjugate which is bound to a situs on theelement separated from the contact portion, and a second receptorcapable of binding the antibody for the analyte, which is bound to theelement between the first receptor and the contact portion. The testsolution moves along the element by capillary action. The situs isexamined for the presence of conjugate, either by exposing the situs toa signal producing means capable of interacting with the label toproduce a signal in a separate development step, such as anenzyme-catalyst-substrate system, or by directly measuring the signalfrom a radioactive label.

U.S. Pat. No. 4,939,098 to Suzuki, et al. discloses an immunoassaydevice for simultaneous determination of at least two components in asample. At least two reagents, each of which reacts specifically withone of the components in the sample, are supported in optional places ona development layer. Residual components in the sample which do notreact with the reagent first contacted by the sample continue to bemoved past the place on the development layer where the first reagent issupported. After the movement of the unreacted components past each ofthe reagent places, the amount of the two reaction products still heldin the development layer are measured. Test reagents may be included inliposomes, which are immobilized on the development layer by physicaladsorption or chemical bonding.

In Suzuki, a detectable label substance such as a chelating agent, anenzyme or a fluorescent substance may be enclosed in the liposomes inaddition to the antibody or antigen test reagents for qualitative orquantitative analysis of sample components. The liposomes or other labelsacs are lysed by the antigen-antibody reaction or complementaryactivity, to release label for detection or quantification. Suzukifurther describes an electric measurement method in which the liposomescontain a substance detectable with electrodes. A solution of theliposomes is removed from the development layer, and the amount of thecomponent to be measured is quantified from the amount of signal in theelectrode.

As a result of the complexity of the device and method described inSuzuki, Suzuki's technique is not well-suited for use in the field, orfor use by untrained personnel. High voltage is required for theelectrophoretic separation method, for example.

Immunoassays employing liposomes for signal production are described inU.S. Pat. Nos. 4,874,710 to Piran and 4,703,017 to Campbell. In Piran,the sample containing the analyte is contacted with a binder for theanalyte in the presence of a conjugate of a ligand coupled to a saclysing agent. The ligand may be designed to bind either with the analyteor the binder. Unbound conjugate, which includes a sac lysing agent,comes into contact with immobilized liposomes, which release adetectable marker. Signal from the marker is measured in the aqueousassay medium. The binder and sacs may be placed on different portions ofa solid support, such as a "dip stick" which may be inserted into andwithdrawn from the assay medium.

Campbell discloses an immunoassay for determination of an analyte usinga tracer, such as the analyte labelled with liposome-encapsulatedmarkers. The tracer can be visually determined without instrumentationand without further treatment of the tracer (such as sac lysing). Abinder for at least one of the analyte and the tracer is supported on atest area of a solid support, which is preferably nitrocellulose in theform of a card, test strip, or dipstick. Detection or quantification ofthe signal, e.g., color from a dye, is made in the test area of thedevice. Competitive, sandwich, and inhibition embodiments of the assayare disclosed.

In view of the above-noted deficiencies and complexities of priortechniques for use as rapid, reliable, and simple field assays, the needremains for technology which will accurately detect and determineanalytes such as environmental and food contaminants.

SUMMARY OF THE INVENTION

The present invention relates to a test device for detecting ordetermining an analyte in a test solution. The test device includes anabsorbent material, having a contact portion proximate to one end forcontact with and uptake of the test solution. Positioned away from thefirst end of the absorbent material, there is a measurement portion towhich is non-diffusively bound a receptor for a conjugate of the analyteto be determined and liposomes which encapsulate a detectable marker.The test device further includes a competitive binding portionpositioned between and segregated from the contact and measurementportions. A binding material for the analyte is non-diffusively bound tothe competitive binding portion.

The present invention further provides a method for detecting ordetermining an analyte in a test sample utilizing the test device of theinvention. A solution containing the analyte and a conjugate of theanalyte and liposomes encapsulating a detectable marker is contactedwith a contact portion proximate to one end of the absorbent material ofthe test device. The test solution is allowed to traverse the absorbentmaterial, via capillary action, from the contact portion through ameasurement portion of the absorbent material which is positioned awayfrom the end to which the contact portion is proximate. The measurementportion has a receptor for the analyte-liposome conjugatenon-diffusively bound thereto. As described above, the absorbentmaterial of the test device further includes a competitive bindingportion between the contact and measurement portions, to which a bindingmaterial for the analyte is non-diffusively bound. After the testsolution has traversed the absorbent material of the test device asdescribed above, the amount of marker in the measurement portion of theabsorbent material, i.e., either the absolute concentration, or theamount relative to some standard reference concentration(s), isdetermined as a measure of the analyte in the sample.

The invention further provides a method and device for determining ananalyte in a test sample employing an automatic electrochemical signalproduction and amplification method. In this aspect, the test devicecomprises an absorbent material, having contact and competitive bindingportions as described above. However, the measurement portion describedabove is replaced in this embodiment with an electrochemical measurementortion. The electrochemical measurement portion may be designed foreither amperometric or potentiometric measurement.

For amperometric measurement, the electrochemical measurement portionhas working, reference, and counter electrode portions, each of which issegregated from each other and from the other portions on the absorbentmaterial. The working, reference, and counter electrodes are adapted forelectrical connection with one another through an appropriateelectrochemical analyzer. Of the three electrode portions, the workingelectrode portion is positioned most adjacent to the competitive bindingportion, and the reference electrode portion is positioned between theworking and counter electrode portions on the absorbent material. Aliposome lysing agent is also non-diffusively bound to the absorbentmaterial, either in the working electrode portion, or in a liposomelysing portion positioned between the competitive binding portion andthe working electrode portion, and segregated from the competitivebinding portion.

The present invention further provides a method for detecting ordetermining an analyte in a test sample utilizing a test device whichrelies on electrochemical detection of an electroactive marker. Anelectrolyte solution containing the analyte and a conjugate of theanalyte and liposomes encapsulating an electroactive marker is contactedwith a contact portion proximate to one end of the absorbent material ofthe test device. The test solution is allowed to traverse the absorbentmaterial, via capillary action, from the contact portion, through anelectrochemical measurement portion of the absorbent material which ispositioned away from the end to which the contact portion is proximate.The electrochemical measurement portion includes working, reference, andcounter electrode portions, each of which is segregated from each otherand from the other portions on the absorbent material. The workingelectrode portion, as described above, is position most adjacent thecompetitive binding portion, with the reference electrode portionpositioned between the working and counter electrode portions on theabsorbent material. The absorbent material further has a liposome lysingagent non-diffusively bound to the absorbent material, either in theworking electrode portion, or in a liposome lysing portion positionbetween the competitive binding portion and the working electrodeportion. The liposome lysing portion is segregated from the competitivebinding portion. As the test solution traverses the absorbent materialof the test device as described above, the flow of electrolyte testsolution through the working electrode and reference electrode portionsand into the counter electrode portion completes a circuit between thecounter and working electrode portions, causing current to flow. Also,the liposomes come into contact with the liposome lysing agent, andlysis of the liposomes causes release of the electroactive marker. Thecurrent flowing between the counter and working electrode portions isthen measured as a measure of the analyte in the sample.

A test device designed for potentiometric marker measurement is alsoprovided in accordance with the invention. This test device is asdescribed above, except that the electrochemical measurement portion hasindicator electrode and reference electrode portions adapted forelectrical contact with one another, wherein the indicator electrodeportion is positioned between and segregated from the competitivebinding portion and from the reference electrode on the absorbentmaterial. A liposome lysing agent is also non-diffusively bound to theabsorbent material, either in the indicator electrode portion, or in aliposome lysing portion which is positioned between the competitivebinding portion and the indicator electrode portion, and which issegregated from the competitive binding portion.

A method for detecting or determining an analyte using such a testdevice is also provided in accordance with the invention. As before, anelectrolyte solution containing the analyte and a conjugate of theanalyte and liposomes encapsulating a electroactive marker is allowed totraverse the absorbent material from the contact portion through theelectroactive measurement portion. As the electrolyte test solutionflows through the indicator electrode portion into the referenceelectrode portion, a potential differential is set up between the twoelectrode portions. In addition, the liposomes are lysed by contact withthe liposome lysing agent. The potential difference between the twoelectrodes is then measured as a measure of the analyte in a sample.

The device and method of the invention can be used directly in thefield. The device is used only once, and, therefore, is free fromresidual environmental contaminants other than what may be present inthe sample to be measured. Samples can be assayed within minutes aftercollection, with the results immediately available on-site. In addition,the device and method of the invention are much less complex than manyof the prior materials and methods. For example, a visible dye can beused as the encapsulated marker, eliminating the need for any detectionor measurement instrumentation, and a separate marker or indicatordevelopment step is not required with any embodiment of the invention.Also, marker-loaded liposomes as used in the device and method of theinvention provide a highly sensitive, rapid or even instantaneous signalproduction/amplification system. Furthermore, the amount ofliposome-encapsulated marker measured in the measurement portion of theabsorbent material of the test device is directly proportional to theanalyte concentration in the sample. This feature of the inventionprovides a particular advantage over prior test devices andimmunoassays, providing an intuitive correlation between signal strengthand analyte concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a multiple channel test device in accordancewith the invention.

FIG. 2 is a schematic of an alternative multiple channel test device inaccordance with the invention.

FIG. 3 is a schematic of the test device of FIG. 1, modified forelectrochemical detection or determination of an analyte.

FIG. 4 is a schematic of a cross-section of a commercially useful testdevice in accordance with the invention.

FIG. 5 is a plot of dose-response data obtained for samples containingvarious concentrations of Alachlor, as described in greater detail inExample 2, below.

FIG. 6 is a schematic of an alternatively modified electrochemical testdevice.

FIG. 7 is a schematic of yet another test device suitable forelectrochemical detection or determination of an analyte.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the present invention is directed to a test devicefor detecting or determining an analyte in a test solution. The testdevice includes an absorbent material which comprises a contact portionproximate to a first end of the absorbent material for contact with anduptake of the test solution. The absorbent material of the test devicefurther comprises a measurement portion at a location on the absorbentmaterial which is positioned away from the first end. The measurementportion has a receptor for a conjugate of the analyte and liposomesnon-diffusively bound thereto, wherein the liposomes have a detectablemarker in the interior thereof. The test device also further comprises acompetitive binding portion positioned between and segregated from thecontact and measurement portions, which competitive binding portion hasa binding material for the analyte non-diffusively bound thereto.

The invention is also directed to a method for detecting or determiningan analyte, including the steps of providing a test device as describedimmediately above, contacting a solution of the analyte and theconjugate with the contact portion of the test device, allowing thesolution to migrate from the contact portion through the measurementportion of the absorbent material, and determining the amount of themarker in the measurement portion as a measure of the analyte in asample.

The invention further provides a method and device for determining ananalyte in a test sample employing an automatic electrochemical signalproduction and amplification method. In this aspect, the test devicecomprises an absorbent material, having contact and competitive bindingportions as described above. However, the measurement portion describedabove is replaced in this embodiment with an electrochemical measurementportion. The electrochemical measurement portion may be designed foreither amperometric or potentiometric measurement.

For amperometric measurement, the electrochemical measurement portionhas working, reference, and counter electrode portions, each of which issegregated from each other and from the other portions on the absorbentmaterial. The working, reference, and counter electrodes are adapted forelectrical contact with one another. Of the three electrode portions,the working electrode portion is positioned most adjacent to thecompetitive binding portion, and the reference electrode portion ispositioned between the working and counter electrode portions on theabsorbent material. A liposome lysing agent is also non-diffusivelybound to the absorbent material, either in the working electrodeportion, or in a liposome lysing portion positioned on the absorbentmaterial between the competitive binding portion and the workingelectrode portion, and segregated from the competitive binding portion.

The present invention further provides a method for detecting ordetermining an analyte in a test sample utilizing a test device whichrelies on electrochemical (amperometric) detection of an electroactivemarker. An electrolyte solution containing the analyte and a conjugateof the analyte and liposomes encapsulating an electroactive marker iscontacted with a contact portion proximate to one end of the absorbentmaterial of the test device. The test solution is allowed to traversethe absorbent material, via capillary action, from the contact portionthrough an electrochemical measurement portion of the absorbent materialwhich is positioned away from the end to which the contact portion isproximate. The electrochemical measurement portion includes working,reference, and counter electrode portions, each of which is segregatedfrom each other and from the other portions on the absorbent material.The working electrode portion, as described above, is position mostadjacent the competitive binding portion, with the reference electrodeportion positioned between the working and counter electrode portions onthe absorbent material. The absorbent material further has a liposomelysing agent non-diffusively bound to the absorbent material, either onthe working electrode portion, or in a liposome lysing portion positionbetween the competitive binding portion and the working electrodeportion. The liposome lysing portion is segregated from the competitivebinding portion. As the test solution traverses the absorbent materialof the test device as described above, the flow of electrolyte testsolution through the working electrode and reference electrode portionsand into the counter electrode portion completes a circuit between thecounter and working electrode portions, causing current to flow. Also,the liposomes come into contact with the liposome lysing agent, andlysis of the liposomes causes release of the electroactive marker. Thecurrent flowing between the counter and working electrode portions isthen measured as a measure of the analyte in the sample.

A test device designed for potentiometric marker measurement is alsoprovided in accordance with the invention. This test device is asdescribed above, except that the electrochemical measurement portion hasindicator electrode and reference electrode portions adapted forelectrial connection with one another, wherein the indicator electrodeportion is positioned between and segregated from the competitivebinding portion and from the reference electrode on the absorbentmaterial. A liposome lysing agent is also non-diffusively bound to theabsorbent material, either in the indicator electrode portion, or in aliposome lysing portion which is positioned between the competitivebinding portion and the indicator electrode portion, and which issegregated from the competitive binding portion.

A method for detecting or determining an analyte using such a testdevice is also provided in accordance with the invention. As before, anelectrolyte solution containing the analyte and a conjugate of theanalyte and liposomes encapsulating an electroactive marker is allowedto traverse the absorbent material through the electroactive measurementportion. As the electrolyte test solution flows through the indicatorelectrode portion into the reference electrode portion, a potentialdifferential is established between the two electrode portions. Inaddition, the liposomes are lysed by contact with the liposome lysingagent. The potential difference between the two electrodes is thenmeasured as a measure of the analyte in a sample.

By "analyte" is meant the compound or composition to be measured that iscapable of binding specifically to an antibody, usually an antigen orhapten.

By "binding material" is meant an immunoglobulin or derivative orfragment thereof having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule--in thiscase, the analyte. The binding material, such as an antibody, can bemonoclonal or polyclonal and can be prepared by techniques that are wellknown in the art such as immunization of a host and collection of seraor hybrid cell line technology. The binding material may also be anynaturally occurring or synthetic compound that specifically binds theanalyte of interest.

By "receptor" is meant any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., egg white avidin, streptavidinthyroxine binding globulin, antibodies, Fab fragments, lectins, nucleicacids, protein A, protein G, and the like.

By "marker accumulating agent" is meant any ion, compound, orcomposition capable of trapping electroactive marker materials releasedfrom the liposome interiors. Ion-exchange resins are preferred markeraccumulating agents in accordance with the invention.

By "absorbent material" is meant a porous material having a pore size offrom 0.05 μm to 50 μm, preferably from 0.45 μm to 5 μm, which issusceptible to traversal by an aqueous medium in response to capillaryforce. Such materials may be natural polymeric materials, particularlycellulosic materials, such as fiber containing papers, e.g., filterpaper, chromatographic paper, etc.; synthetic or modified naturallyoccurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, activated nylon, etc.; either used by themselves or inconjunction with a support, as described below.

The absorbent material may be polyfunctional or be capable of beingpolyfunctionalized to permit covalent bonding of receptors or antibodiesas well as to permit bonding of other compounds which form a part of thesignal producing system.

The absorbent material which is employed in the test device and methodof the invention is generally a cellulose ester with nitrocellulosegiving exceptionally good results. It is to be understood that the term"nitrocellulose" refers to nitric acid esters of cellulose, which may benitrocellulose alone, or a mixed ester of nitric acid and other acids,and in particular, aliphatic carboxylic acids having from one to sevencarbon atoms, with acetic acid being preferred. Such materials, whichare formed from cellulose esterified with nitric acid alone, or amixture of nitric acid and another acid such as acetic acid, are oftenreferred to as nitrocellulose paper.

Although nitrocellulose is a preferred material for producing the testdevice, it is to be understood that other materials, having a surfacearea sufficient for supporting the binding material in a concentrationas hereinbelow described may also be employed for producing such testdevices.

In general, the absorbent material which is used in the device andmethod of the invention has a surface area such that is capable ofsupporting the binding material in a concentration of at least 1 μg/cm²,(most generally in a concentration of least 10 μg/cm²) and preferably atleast 40 μg/cm².

Absorbent materials having high surface areas (such as nitrocellulose)are particularly preferred in that the binding material may be supportedon such materials in a high concentration. It is to be understood,however, that the concentration of binding material which is actuallyused is dependent in part on the binding affinity of the bindingmaterial. Accordingly, the scope of the invention is not limited to aparticular concentration of binding material on the absorbent material.

Application of receptors, binding materials, liposome lysing agents, andmarker accumulating agents to the absorbent material may be accomplishedby well-known techniques, for example, by spraying or spotting asolution of those materials onto the absorbent material.

The amount of receptor which is bound to the absorbent material at themeasurement portion will vary depending upon the amount required to bindthe unbound conjugate to enable an effective assay. Generally, theamount of receptor at the measurement portion will be at least 10μg/cm².

The receptor and the binding material and, where desired, members of thesignal producing system, can be bound to the absorbent material byadsorption, rather than covalent bonding, as long as such binding isnon-diffusive. This will involve contacting the absorbent material witha solution containing the materials to be bound to the material andallowing the material to dry. In general, this procedure will be usefulonly where the absorbent material is relatively hydrophobic or has ahigh surface charge, and subsequent treatment with proteins, detergents,polysaccharides, or other materials capable of blocking non-specificbinding sites will be required.

After application of the binding material to the competitive bindingportion on the absorbent material, the residual binding capacity of theabsorbent material is saturated or blocked with one or more types ofproteins or other compounds such as polyvinylpyrrolidone,polyvinylalcohol, etc. which do not specifically bind the materials tobe employed in the assay. Thus, for example, the residual bindingcapacity of the substrate may be blocked so as to prevent non-specificbinding by the use of bovine serum albumin, as described in Towbin, etal., Proc. Nat'l. Acad. Sci., 76 (1979) 4350, which is herebyincorporated by reference. The techniques for preventing non-specificbinding are generally known in the art, and such techniques are alsogenerally applicable to reventing non-specific binding in the assay ofthe present invention. Examples of particularly suitable techniques forblocking with polyvinylpyrrolidone and polyvinylalcohol are described,for example, Bartles, et al. Anal. Biochem., 140 (1984) 784, and inBritish Patent Specification GB 2204398 A, respectively, which arehereby incorporated by reference.

In conjunction with a blocking agent, a surfactant may be applied to theabsorbent material in a concentration sufficient to facilitate migrationof the analyte-liposome conjugate without lysis of the liposomes.

The absorbent material can be a single structure such as a sheet cutinto strips. The absorbent material can be mounted on a supportmaterial. On the other hand, the absorbent material may provide its ownsupport. In one embodiment of the invention, the test device is a stripof particulate material bound to a support or solid surface such asfound, for example, in thin-layer chromatography. The absorbent materialcan be a sheet having lanes thereon, or be a uniform sheet capable ofdivision into separate lanes by physical removal of the absorbentmaterial from the support to induce lane formation, wherein a separateassay can be performed in each lane as shown in FIGS. 1-3 and 6-7. Theabsorbent material can have a shape that is rectangular, circular, oval,triagonal, or the like, provided that there is at least one direction oftraversal of a test solution by capillary migration. Other directions oftraversal may occur such as in an oval or circular piece contacted inthe center with the test solution. However, the main consideration isthat there e one direction of flow from the contact portion through themeasurement portion. In this discussion strips of absorbent material aredescribed by way of illustration and not limitation.

FIG. 1 is a schematic of a test device in ccordance with the invention,depicted immediately after nsertion into control solution 209 and testsolution 208, hich are held in tray 210 having partition 216 extendingacross the entire width of tray 210 to divide tray 210 into separatecompartments for the control and test solutions. As shown in FIG. 1,absorbent material 212 is mounted on support 214. The test device shownin FIG. 1 is divided into two channels, namely, control channel D andtest channel E, and competitive binding portions 204 and measurementportions 206. Control channel D includes competitive binding portion204d, which has a binding material for the analyte of interestnon-diffusively bound thereto. Control channel D further includesmeasurement portion 206d, which, as described above, has a receptor forthe appropriate analyte-liposome conjugate non-diffusively boundthereto. Test channel E similarly has competitive binding portion 204eand measurement portion 206e, which have been constructed to recognizeand bind the analyte and the analyte-liposome conjugate, respectively,as described above.

According to the embodiment of the invention shown in FIG. 1, thecontact portion of each channel of the test strip is the end of thestrip to be inserted into the test or control solutions.

Test solution 208 is typically prepared, as described below, bycombining a sample known or suspected to contain the analyte with theanalyte-liposome conjugate in an aqueous medium. In accordance with theembodiment shown in FIG. 1, control solution 209 is similarly preparedto have the same concentration of the conjugate as test solution 208,and a known concentration of analyte.

In use, the contact portion of absorbent material 212 of control channelD is inserted into control solution 209, while the contact portion ofabsorbent material 212 of test channel E is inserted into test solution208. Wetting of absorbent material 212 by capillary action is allowed tocontinue at least until measurement portions 206d and 206e are wet withcontrol solution 209 and test solution 208, respectively. As controlsolution 209 and test solution 208 traverse channels D and E throughcompetitive binding portions 204d and 204e, the analyte in controlsolution 209 and test solution 208 competes with the analyte-liposomeconjugate in each of the solutions for available binding sites on thespecific binding material bound to competitive binding portions 204d and204e. Control solution 209 and test solution 208 continue to traversechannels D and E of the test device into and through measurementportions 206d and 206e, where the conjugate is trapped and accumulatedin measurement portions 206d and 206e by the specific conjugate receptorbound thereto.

As is described below, qualitative measurement of the marker inmeasurement zones 206d and 206e may be made visually when the marker isa visible dye. Alternatively, the intensity of the color in measurementportions 206d and 206e may be visually compared with a series ofreference standards, such as in a color chart, for a semi-quantitativedetermination of the amount of analyte in the sample. Other types ofmarkers, as described below, may be detected and measured and usinginstrumentation such as a spectrophotometer or fluorimeter.

An alternative multiple channel test device in accordance with theinvention is shown schematically in FIG. 2. In this case, the testdevice is shown before insertion into sample compartment 110 containinga wicking reagent such as carrier solution 108, which is generally abuffered saline solution. In the embodiment shown in FIG. 2, the testdevice is divided into high control channel A, test channel B, and lowcontrol channel C. As was described above in connection with FIG. 1,absorbent material 112 is supported on support 114. The test deviceincludes wicking portions 100, contact portions 102, competitive bindingportions 104, and measurement portions 106. High control channel Aincludes contact portion 102a, competitive binding portion 104a, andmeasurement portion 106a. Test channel B similarly includes contactportion 102b, competitive binding portion 104b, and measurement portion106b. Finally, low control channel C includes contact portion 102c,competitive binding portion 104c, and measurement portion 106c.

The test device shown schematically in FIG. 2 is designed for thesimultaneous measurement of the analyte in a test sample and high- andlow-level control compositions to provide linear interpolation andverification of response. A high-level control solution, the testsolution, and a low-level control solution are spotted onto contactportions 102a, 102b, and 102c, respectively, prior to insertion of thetest device into carrier solution 108, which includes theanalyte-liposome conjugate. Following migration of carrier solution 108through contact portions 102a, 102b, and 102c, competitive bindingportions 104a, 104b, and 104c, and measurement portions 106a, 106b, and106c and, optionally, to the end of channels A, B and C, color intensityor other marker signal is observed or quantified in measurement zones106a, 106b, and 106c which, as described above in connection with FIG.1, each have the receptor for the analyte-liposome conjugatenon-diffusively bound thereto. As was also described in connection withFIG. 1, the analyte in each of the high- and low-level controlsolutions, and the test solution, and the analyte-liposome conjugate inthe carrier solution, compete for available binding sites on the bindingmaterial, which is non-diffusively bound to competitive binding portions104a, 104c, and 104b, respectively.

The support for the absorbent material where a support is desired ornecessary will normally be hydrophobic, water insoluble, non-porous, andrigid, and usually will be of the same length and width as the absorbentstrip but may be larger or smaller. A wide variety of organic andinorganic materials, both natural and synthetic, and combinationsthereof, may be employed, provided only that the support does notinterfere with the production of signal from the marker. Illustrativepolymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylonpoly(vinyl butyrate), glass, ceramics, metals, and the like.

The size of the piece of absorbent material is dependent on severalconsiderations. The primary consideration is to separate unboundconjugate from bound conjugate and to capture unbound conjugate at themeasurement portion to give a sufficient signal so that a sensitive andaccurate assay is achieved. The following discussion is primarilyfocused on strips of absorbent material for purpose of illustration andnot limitation. As mentioned above, other shapes such as circular, oval,triagonal, and the like, fall equally within the scope of thisinvention. The dimensions thereof and other parameters can be determinedby those skilled in the art with reference to the disclosure herein.

When capillary flow is predominantly upward, the length and thickness ofthe strip control the amount of solution that can pass through themeasurement portion. If the transfer of a large volume of test solutionis desired, the fluid capacity of the strip above the measurementportion must be sufficient to accommodate the desired volume.Alternatively, an absorbing pad may be used to contact the end of thestrip opposite the end used to contact the test solution.

To permit conservation of reagents and provide for samples of limitedsize, the width of the strip will generally be relatively narrow,usually less than 20 mm preferably less than 10 mm. Generally, the widthof the strip will not be less than about 2 mm and will usually rangefrom about 2 mm to 10 mm, preferably from about 3 mm to 6 mm.

As is described in detail below, the test device in accordance with theinvention may be modified for simultaneous multiple analyte detection ordetermination. The length of the strip will depend on the concentrationof the analyte and practical considerations such as ease of handling andthe number of measurement portions on the strip and will be about 4 cmto 20 cm, usually about 5 cm to 15 cm, preferably about 6 to 13 cm butmay be of any practical length. The structure of the strip can be variedwidely and includes fine, medium fine, medium, medium coarse and coarse.In general, smaller pore size and finer material will provide slowcapillary flow and more efficient capture of bound conjugate on thestrip. Courser, more porous materials provide faster flow, but theefficiency of capture is reduced. Selection of the porosity of thematerial depends on the rate of binding of the components for a givenassay.

The position of the competitive binding portion, and measurement portion(or portions, where a plurality of analytes are being determined),should be governed by the basic principle involved in the presentinvention. One desires to pass by capillarity a sufficient amount of thetest solution through the strip to the measurement portion to separatebound conjugate from unbound conjugate and to bind the unbound conjugateat the measurement portion to produce a signal that is detectable. It isdesirable to position the measurement portion close to the competitivebinding portion. Desirably, the measurement portion should be at least 3mm, preferably at least 8 mm, from the competitive binding portion ofthe strip. The measurement portion should be positioned on the absorbentmaterial so as to enable the test solution to pass through themeasurement portion by capillary action so as to capture the unboundconjugate. Generally, the distance between the competitive bindingportion and the contact portion should be at least 2 mm, preferably atleast 5 mm. Where several measurement portions are used formulti-analyte determinations, the measurement portions can be groupedclose together or apart but must not be so close as to compromiseresolution of the signals. Consequently, such measurement portionsusually should be spaced not less than 0.5 mm apart, preferably at least1 mm apart.

In carrying out the method of the invention, the protocol will normallyinvolve combining the sample suspected of containing the analyte withthe conjugate in an aqueous medium to form the aqueous test solution.The sample may be derived from a wide variety of sources, such asphysiologic fluids, illustrated by saliva, serum, plasma, urine, ocularlens fluid, spinal fluid, etc., chemical processing streams, food, wastewater, natural waters, soil extracts, etc. Various addenda may be addedto adjust the properties of the test solution, or of a carrier solutionused as a wicking reagent, depending upon the properties of the othercomponents of the device, as well as on those of the liposomes or theanalyte-liposome conjugate, or the analyte itself. Examples of solutionaddenda which may be incorporated into test, control, or carriersolutions in accordance with the invention include buffers, and sampleor analyte solubilizing agents, such as, for example, nonpolar solvents.

The contact portion of the absorbent material, which usually includesthe end of the absorbent material to which the contact portion isproximate, is contacted with test solution, usually by immersion of thecontact portion into the test solution. Wetting of the absorbentmaterial by capillary action is allowed to continue at least until themeasurement portion is wet.

Alternatively, the test solution may be contacted with the absorbentmaterial by spotting the test solution onto the absorbent material inthe contact portion. In this case, the contact portion includes awicking portion at the first end of the absorbent material. In use, thewicking portion of the contact portion is inserted into a wickingreagent after the test solution is spotted onto the contact portion,outside of the wicking portion.

For the most part, relatively short times are involved for the testsolution to traverse the strip. Usually, traversal of the test solutionover the strip will take at least 30 seconds and not more than 1/2 hour,more usually from about 1 minute to 10 minutes. In accordance with themethod of the invention, the signal is rapidly, even immediately,detectable.

The conjugate of the analyte and the marker-encapsulating liposome maybe prepared by procedures generally known in the art, with theparticular procedure used in a given case being dependent upon theanalyte which is employed. Such techniques include covalent coupling,derivatization or activation, and the like. The liposomes may beproduced from a component which has been derivatized with the analyte,whereby the liposomes, when produced, are conjugated with the analyte.In another procedure, the liposomes, including the marker, may beinitially formed, followed by conjugating the liposomes with analyte byprocedures known in the art.

Liposomes can be prepared from a wide variety of lipids, includingphospholipids, glycolipids, steroids, relatively long chain alkylesters; e.g., alkyl phosphates, fatty acid esters; e.g. lecithin, fattyamines, and the like. A mixture of fatty materials may be employed, suchas a combination of neutral steroid, a charge amphiphile and aphospholipid. Illustrative examples of phospholipids include lecithin,sphingomyelin, and dipalmitoylphosphatidylcholine, etc. Representativesteroids include cholesterol, chlorestanol, lanosterol, and the like.Representative charge amphiphilic compounds generally contain from 12 to30 carbon atoms. Mono- or dialkyl phosphate esters, or alkyamines; e.g.dicetyl phosphate, stearyl amine, hexadecyl amine, dilaurylphosphate,and the like are representative.

The liposome sacs are prepared in aqueous solution containing the markerwhereby the sacs will include the marker in their interiors. Theliposome sacs may be prepared by vigorous agitation in the solution,followed by removal of the unencapsulated marker. Further details withrespect to the preparation of liposomes are set forth in U.S. Pat. No.4,342,826 and PCT International Publication No. WO80/01515, both ofwhich are incorporated by reference.

As hereinabove indicated, the signal producing system includes a markerincluded in the interior of the conjugated liposomes. Suitable markersinclude fluorescent dyes, visible dyes, bio- and chemiluminescentmaterials, enzymatic substrates, and radioactive materials. Visible dyesand radioactive materials can be measured without lysis of theliposomes. However, even when liposome lysis is required, as when theother marker materials are used, a separate lysing step is notnecessary, because a liposome lysing agent may be non-diffusively bounddirectly to the absorbent material as, for example, in the measurementzone. Suitable liposome lysing materials include surfactants such asoctylglucopyranoside, sodium dioxycholate, sodium dodicylsulfonate,polyoxyethylenesorbitan monolaurate sold by Sigma under the trademarkTween-20, and a non-ionic surfactant sold by Sigma under the trademarkTriton X-100, which is t-octylphenoxypolyethoxyethanol. Alternatively,complement lysis of liposomes may be employed.

A qualitative or semi-quantitative measurement of the presence or amountof an analyte of interest may be made with the unaided eye when visibledyes are used as the marker. Alternatively, when greater precision isdesired, or when the marker used necessitates instrumental analysis, theintensity of the marker may be measured directly on the absorbentmaterial using a quantitative instrument such as a fluorimeter,spectrophotometer, etc.

In one embodiment of the invention, a marker which is visible under theassay conditions is used so that the presence and/or amount of analytemay be determined without further treatment and without the use ofinstrumentation, e.g., by use of a liposome containing a dye as themarker.

In the method of the invention, a conjugate of the analyte andmarker-loaded liposomes label are combined in an aqueous medium with asample suspected of containing the analyte, to provide an aqueous testsolution. Alternatively, the combination of the conjugate and the samplesuspected of containing the analyte can take place on the absorbentmaterial. The primary consideration is that a test solution containingthe sample come in contact with a conjugate of the analyte and themarker-loaded liposomes prior to or at the contact portion of theabsorbent material. A receptor capable of binding to the conjugate isnon-diffusively bound to the absorbent material at the measurementportion. The binding material is non-diffusively bound to the absorbentmaterial between the measurement portion and the contact portion. Thecontact portion of the absorbent material is contacted with the testsolution, which will traverse the absorbent material through capillaryaction. This transversal can be upward, downward, horizontal orcombinations thereof. The amount of the conjugate that becomes bound tothe measurement portion through binding to the receptor is related tothe amount of analyte in the sample. The signal producing systemprovides a detectable signal at the measurement portion only when theconjugate is bound to the receptor in the measurement portion, so thatthe presence of the analyte may be determined by detecting the signal atthe measurement portion. Binding of the conjugate to the receptor mayoccur directly to a binding site on the liposome.

The present invention provides for an immunoseparation of boundconjugate from unbound conjugate. This is accomplished by having thebinding material receptor non-diffusively bound to the absorbentmaterial in the competitive binding portion between the measurementportion and the contact portion. A binding material will normally bechosen that provides for direct binding to the analyte. Usually, thebinding material will be present in an amount that will provide theappropriate sensitivity required for a specific analyte.

The movement of the test solution along the absorbent material is due tocapillary action. This capillary movement along the absorbent materialcauses the test solution to be carried to and through the measurementportion.

Measurement of the marker-loaded liposomes takes place in themeasurement portion of the absorbent material. As described above,concentration or accumulation of the conjugate may be achieved byvarious immunospecific binding reactions as described above.

In one embodiment of the invention, the conjugate of the analyte and theliposomes is further bound to biotin. The assay is carried out in thesame way but the receptor is anti-biotin such as avidin or antibody forbiotin. When analyte is present, some biotinylated conjugate reaches themeasurement portion and is bound by the anti-biotin or avidin. However,it has been found that egg white avidin, with the carbohydrate moietystill attached, strongly binds all of the liposomes, without the need ofconjugating biotin to them. As the specificity of the assay lies in theimmunorecognition reaction in the antibody zone, an avidin collectionzone provides a simple solution to give the desired direct readoutmeasurement.

In an electrochemical detection method, an electroactive species, suchas ferrocyanide, is encapsulated into the liposomes. Electrodes areprinted onto the strip, or the strip is placed in contact with theelectrodes. After lysis of the liposomes, the quantity of theelectroactive species is determined amperometrically orpotentiometrically.

FIG. 3 is a schematic of a test device in accordance with the inventionwhich employs electrochemical detection or measurement of anelectroactive marker. The device in FIG. 3 is shown immediately afterinsertion into control solution 309 and test solution 308, which arekept separated from one another by partition 316 in tray 310. As wasdescribed above in connection with FIG. 1, the device is divided intocontrol channel F and test channel G and includes absorbent material 312mounted on support 314. As is the case with the device shown in FIGS. 1and 2, competitive binding portion 304f on absorbent material 312 incontrol channel F and competitive binding portion 304g on absorbentmaterial 312 in test channel G are substantially identical to oneanother prior to contact of control solution 309 and test solution 308with those portions, respectively. Each of the competitive bindingportions has the same binding material for the analyte of interestnon-diffusively bound thereto. The test device shown in FIG. 3 furtherincludes working electrode portions 318f and 318g for control channel Fand test channel G, respectively. Reference electrode portions 320f and320g and counter electrode portions 322f and 322g are also included onabsorbent material 312 of the test device, as shown in FIG. 3.

Other than the working, reference, and counter electrode portions shownin FIG. 3, the test device is constructed as described above inconnection with FIG. 1 and 2. Each of the electrode portions isseparated from the others on the absorbent material. Working electrodeportion 318f, reference electrode portion 320f, and counter electrodeportion 322f are each adapted for electrical connection to one anothervia connections 332, 334, and 336, respectively, to potentiostat 338.Working electrode portion 318g, reference electrode portion 320g, andcounter electrode portion 322g are similarly each adapted for electricalconnection to one another via connections 324, 326, and 328,respectively, to potentiostat 330. In operation, the working, reference,and counter electrode portions 318g, 320g, and 322g of channel G are inelectrical contact with one another, as is the case with referenceelectrode and working electrode portions 318f, 320f, and 322f on channelF.

Reference electrodes 320f and 320g will usually be silver electrodes,while working electrode portions 318f and 318g, and counter electrodeportions 322f and 322g may be prepared from any suitable materials suchas the noble metals, other metals such copper and zinc, or carbonelectrode materials in various forms, including graphitic, glassy andreticulated carbon materials.

Counter electrodes 322f and 322g may be composed of the same or adifferent material from working electrodes 318f or 318g.

Each of the electrode portions shown in FIG. 3 may be prepared by screenprinting of the electrode materials onto absorbent material 312. As iswell known, screen printing involves preparation of an organic oraqueous slurry of the electrode material, typically, a fine powder ofcarbon, gold, etc., followed by application of the slurry across andthrough a silk screen onto the absorbent material of the test device.This slurry may optionally include a polymeric binder which aids inaggregating the fine metallic particles together on the surface of theabsorbent material. The electrode material slurry may be fixed on thesurface of the absorbent material by heating, however, the printedelectrode portions are preferably allowed to air dry on the surface ofthe absorbent material.

The test device shown in FIG. 3 is designed for amperometric detectionor quantification of an electroactive marker included in the interior ofthe liposomes included in the analyte-liposome conjugate incorporated incontrol solution 309 and test solution 308. Following insertion of thetest device into control solution 309 and test solution 308, as shown inFIG. 3, and as described above in connection with FIG. 1, the controland test solutions are allowed to traverse the device, from the contactportions of both channels, through counter electrode portions 322f and322g. Competitive binding between the analyte and analyte-liposomeconjugate occurs in competitive binding portions 304.

In the embodiment of the invention shown in FIG. 3, working electrodeportions 318f and 318g include a marker accumulating agent, such asanion-exchange polymer, non-diffusively bound thereto. Working electrodeportions 318f and 318g further incorporate a liposome lysing agent, asdefined above, in an amount sufficient to lyse all of the liposomescontacting the lysing agent.

Alternatively, the liposome lysing agent may be non-diffusively bound toabsorbent material 312 in liposome lysing portions (not shown) locatedon absorbent material 312 in each of channels F and G betweencompetitive binding portion 304f and working electrode portion 318f inchannel F, and between competitive binding portion 304g and workingelectrode portion 318g on channel G. The liposome lysing portions mustbe separate from competitive binding portions 304f and 304g on absorbentmaterial 312.

As control solution 309 and test solution 308, which are electrolytesolutions such as saline solutions of the analyte and analyte-liposomeconjugate, traverse channels F and G of the test device through workingelectrode portions 318f and 318g, the liposomes in the conjugate arelysed immediately before contact with or upon entry into workingelectrode portions 318f and 318g to release an electroactive markersubstance included in their interiors. Electroactive markers arematerials capable of undergoing oxidation or reduction. Suitableelectroactive markers include metal ions, and organic compounds such asascorbate, ascorbic acid, quinones, phenols, NADH. Ferrocyanide is themost preferred electroactive marker in accordance with the invention.

The electroactive marker released from the interior of the liposomesincorporated in the analyte-liposome conjugates in control solution 309and test solution 308 are then accumulated by the ion-exchange materialbound to working electrode portions 318f and 318g throughout the periodduring which the control and test electrolyte solutions migrate pastreference electrode portions 320f and 320g to counter electrode portions322f and 322g. At that point, the electrical circuits between theworking, reference, and counter electrode portions of each of channels Fand G is automatically completed and electrolysis of the accumulatedelectroactive marker occurs. The current flowing through the circuits,which is directly proportional to the amount of marker released by theliposomes, and corresponds to the amount of analyte in the sample, isthen measured by potentiostat 330. Devices which may be used aspotentiostats in accordance with the invention include the CypressSystem Electrochemical Analyzer and the BAS Electrochemical Analyzer.

An alternative design for a test device constructed for electrochemicaldetection or quantification of a liposome-encapsulated electroactivemarker is shown schematically in FIG. 6. Electrolyte control solution609 and electrolyte test solution 608, separated by a partition 616 intray 610, are as described above in connection with the correspondingfeatures shown in FIG. 3. Similarly, the test device shown in FIG. 6,comprising control channel H and test channel I, each of which comprisesabsorbent material 612 mounted on support 614, and each of which hascomparative binding portions 604, are as described above for thecorresponding structures described above in connection with FIG. 3. Thedevice shown in FIG. 6 further includes working electrode portions 618hand 618i, reference electrode portions 620h and 620i, and counterelectrode portions 622h and 622i, which are separated from one anotheron absorbent material 612. Working electrode portion 618h is adapted forelectrical connection to potentiostat 638, and through potentiostat 638to reference electrode portion 620h and counter electrode portion 622h,via connection 632. Working electrode portion 618i is similarly adaptedfor electrical connection to potentiostat 630, and through potentiostat630 to reference electrode portion 620i and counter electrode portion622i, via connection 624. Similarly, reference electrode portions 620hand 620i, and counter electrode portions 622h and 622i, are adapted forelectrical connection through connections 634 and 626, and throughconnections 636 and 628, respectively, to potentiostats 638 and 630.

The test device shown in FIG. 6 further incorporates a liposome lysingagent non-diffusively bound to a absorbent material 612, either inworking electrode portions 618h and 618i, or, alternatively, in separateliposome lysing portions (not shown) on absorbent material 612 in eachof channels H and I between competitive binding portion 604h and workingelectrode portion 618h, on control channel H, and between competitivebinding portion 604i and working electrode portion 618i on absorbentmaterial 612 of test channel I. As in the case of the device shown inFIG. 3, the separate liposome lysing portions must be separated fromcompetitive binding portions 604h and 604i on absorbent material 612 ineach of channels H and I. However, it is not necessary that the liposomelysing portion be separated from working electrode portions 618h and618i.

In contrast to the device shown in FIG. 3, the device shown in FIG. 6does not include a marker accumulating agent in working electrodeportions 618h and 618i. In operation, traversal of the test device byelectrolyte control solution 609 and electrolyte test solution 610proceeds as described previously in connection with FIG. 3 throughworking electrode portions 618h and 618i, reference electrode portions620h and 620i, and counter electrode portions 622h and 622i, however,electroactive marker released as a result of contact between theanalyte-liposome conjugate and the liposome lysing agent incorporated onabsorbent material 612 in each of control channel H and test channel Iis not accumulated in working electrode portions 618h and 618i. In thisembodiment of the invention, once the electrical circuits betweencounter electrode portions 622h and 622i, and working electrodes 618hand 618i are completed, electrolysis of the released electroactivemarker occurs continuously as the marker flows past working electrodeportions 618h and 618i. The current measured by potentiostats 630 and638 is then integrated for a fixed period of time to provide a measureof the amount of analyte in test solution 608.

Yet another embodiment of the test device of the present inventionemploy electrochemical detection is shown schematically in FIG. 7. Inthis case however, the potentiometric measurement of an electroactivemarker released from the interior of the liposomes comprising theanalyte-liposome conjugate is made. As was the case with the deviceshown in FIG. 6, electrolyte control solution 709 and electrolyte testsolution 708 are kept separated by partition 716 in tray 710. The testdevice shown in FIG. 7 is constructed as was described above inconnection with FIGS. 3 and 6, and includes control channel J and testchannel K, each comprising absorbent material 712 mounted on support714. As was the case with FIGS. 1, 3, and 6 described above, the devicein FIG. 7 is shown immediately after insertion of the contact portion ofeach of channels J and K into control solution 709 and test solution708, respectively.

The device shown in FIG. 7 further includes competitive binding portions704. In the case of the test device shown FIG. 7, however, the devicecomprises indicator electrode portions 718j and 718k, and referenceelectrode portions 720j and 720k, which electrode portions are separatedfrom one another on absorbent material 712, as shown in FIG. 7.Indicator electrode portions 718j and 718k, and reference electrodeportions 720j and 720k are adapted for electrical connection topotentiometers 736 and 730 through connections 732 and 724,respectively, for indicator electrode portions 718j and 718k, and 734and 726, respectively, for reference electrode portions 720j and 720k.

As was described above in connection with FIG. 3, reference electrodeportions 720j and 720k may be prepared, as described above, by screenprinting a slurry of finely divided silver powder onto absorbentmaterial 712. Indicator electrode portions 718j and 718k may be preparedin the same way from any of the electrode materials described above inconnection with working electrode portions 318f and 318g and counterelectrode portions 322f and 322g, of FIG. 3.

It is necessary to incorporate a liposome lysing agent on absorbentmaterial 712 of the test device shown in FIG. 7 between competitivebinding portion 704j and reference electrode portion 720j of channel Jand between competitive binding portion 704k and reference electrodeportion 720k of channel K. However, as was described above in connectionwith FIGS. 3 and 6, the liposome lysing agent may either benon-diffusively bound to absorbent material 712 in indicator electrodeportions 718j and 718k, or may it be bound to a separate liposome lysingportion located on absorbent material 712 between competitive bindingportion 704j and indicator electrode portion 718j in channel J, andbetween competitive binding portion 704k and indicator electrode portion718k in channel K. As before, it is necessary that such liposome lysingportions be separated from competitive binding portions 704 on absorbingmaterial 712 in channels J and K.

Traversal of channel J by electrolyte control solution 709 andsimultaneous traversal of channel K by electrolyte test solution 708,from the contact portions of channels J and K through competitivebinding portions 704, which, as described above, have a binding materialfor the analyte of interest non-diffusively bound thereto, proceeds asdescribed above in connection with FIGS. 3 and 6. As control solution709 and test solution 708 migrate through indicator electrode portions718j and 718k into reference electrode portions 720j and 720k, apotential differential is set up between the indicator and referenceelectrode portions in each channel. These potential differentials aremeasured by potentiometers 736 and 730, which may be pH meters, such asthose available from Orion, Corning, or Beckman. The potentialdifferentials are directly proportional to the concentrations of theelectroactive markers released from the liposome interiors, andcorrespond to the concentrations of the analyte in the control and testsolutions.

It should be noted that although the working, counter, and referenceelectrode portions in FIGS. 3 and 6, and the reference and indicatorelectrodes in FIG. 7 have been shown in specific positions, thepositions can be otherwise than shown. Specifically, for example, therelative positions of the reference and working electrode portions inFIGS. 3 and 6 may be reversed. Similarly, the positions of the referenceand indicator electrodes shown in FIG. 7 may be reversed. Although thecounter electrode portions in FIGS. 3 and 6 will usually be as shownwith respect to the working and reference electrode portions, even theposition of the counter electrode within the electrochemical measurementportion is not critical.

The solvent for the test solution will normally be an aqueous medium,which may be up to about 40 weight percent of other polar solvents,particularly solvents having from 1 to 6, more usually of from 1 to 4,carbon atoms, including alcohols, dimethylformamide anddimethylsulfoxide, dioxane and the like. Usually, the cosolvents will bepresent in less than about 20 weight percent. Under some circumstances,depending on the nature of the sample, some or all of the aqueous mediumcould be provided by the sample itself.

The pH for the medium will usually be in the range of 4-10, usually 5-9,and preferably in the range of about 6-8. The pH is chosen to maintain asignificant level of binding affinity of the binding members and optimalgeneration of signal by the signal producing system. Various buffers maybe used to achieve the desired pH and maintain the pH during the assay.Illustrative buffers include borate, phosphate, carbonate, tris,barbital and the like. The particular buffer employed is not critical,but in individual assays, one buffer may be preferred over another.

Moderate, and desirably substantially constant, temperatures arenormally employed for carrying out the assay. The temperatures for theassay and production of a detectable signal will generally be in therange of about 4°-40° C., more usually in the range of about 10°-35° C.,and frequently, will be ambient temperatures, that is, about 15°-25° C.

The concentration, in the liquid sample, of analyte which may be assayedwill generally vary about 10⁻³ to about 10⁻¹⁵ M, more usually from about10⁻⁵ to 10⁻¹⁰ M. Considerations such as the concentration of the analyteof interest and the protocol will normally determine the concentrationof the other reagents.

With the test device and method of the invention, one may also assay atest solution for a plurality of analytes such as toxic chemicals, orscreen for one or more of a plurality of analytes. In one embodiment,the test device includes multiple measurement portions, each of whichhas a distinctive receptor specific for one of several haptens, whichare chosen, in part, so as not to interfere with any of the analytes ofinterest. The test solution (and control solution), where appropriate isformed by mixing together in an aqueous medium the sample and aplurality of liposome conjugates each of which comprises (a) one of theanalytes and (b) a hapten which will bind specifically to one of thereceptors in one of the measurement portions of the device. Thus, thestrip contains a separate measurement portion for each analyte. Amixture of specific binding materials for each of the analytes isnon-diffusively bound to the absorbent material in a single competitivebinding portion between the contact portion and the measurementportions. The conjugate of each of the analytes to be determined in thisembodiment of the invention, may include a marker which is detectabledistinctly from the other markers. With different encapsulated dyes, theresults of the assay can be "color coded". Alternatively, each analytemay be determined by assignment of each conjugate/analyte to its ownmeasurement portion for concentration and measurement.

In an alternative multiple-analyte embodiment, the measurement portionhas bound thereto separate receptors capable of binding differentanalyte-liposome conjugates through the recognition of the receptors forseparate haptens on the different analyte-liposome conjugates. Usingsuch a device, it is possible to conduct a screening assay to determine,for example, whether any of a group of analytes is present in thesample. Alternatively, the liposomes attached to each analyte can have adifferent dye encapsulated, and a multi-wavelength detector can be usedin a measurement portion, such as an egg-white avidin portion.

As a matter of convenience, the present device can be provided in a kitin packaged combination with predetermined amounts of reagents for usein assaying for an analyte or a plurality of analytes. Aside from theabsorbent test device and the analyte-liposome conjugate, otheradditives such as ancillary reagents may be included, for example,stabilizers, buffers, and the like. The relative amounts of the variousreagents may be varied widely, to provide for concentration in solutionof the reagents which substantially optimizes the sensitivity of theassay. The reagents can be provided as dry powders, usually lyophilized,including excipients, which on dissolution will provide for a reagentsolution having the appropriate concentrations for performing the assay.The kit or package may include other components such as standards of theanalyte or analytes (analyte samples having known concentrations of theanalyte).

FIG. 4 is a schematic of a cross-section of a commercially useful testdevice in accordance with the invention. Absorbent material 420 issupported on strip holder 410, which may be composed of any inert rigidor semi-rigid support material and is preferably composed of plastic. Asdescribed above in connection with FIGS. 1 and 2, absorbent material 420includes contact portion 402, competitive binding portion 422 having abinding material for the analyte of interest non-diffusively boundthereto, and measurement portion 424, having a receptor for a conjugateof the analyte and marker-encapsulating liposomes non-diffusively boundthereto. In the embodiment of the invention shown in FIG. 4, theabsorbent material of the test device also includes endpoint indicatorportion 408, as described below. Compartment covers 414 and 418 providefurther support and protection for absorbent material 420. Covers 414and 418, which are constructed of the same or similar rigid orsemi-rigid support materials as strip holder 410, may, with strip holder410, comprise a single molded piece. Alternatively, covers 414 and 418may be prepared from a transparent material which allows viewing ofabsorbent material 420, and may be connected to strip holder 410.

The device shown in FIG. 4 further include windows 416 which providevisual access to measurement portion 424 and end-point indicator portion408 of the absorbent material. Windows 416 may be made from atransparent material such as plastic or glass. Alternatively, they maybe prepared from an opaque material such as a colored plasticincorporating holes through which absorbent material 420 may be seen.Air vent 426 provides an outlet for air forced out of absorbent material420 as a test solution or control solution migrates along absorbentmaterial 420 from contact portion 402 to end-point indicator portion408.

The embodiment shown in FIG. 4 provides a sturdy, portable,contamination-resistant test device suitable for use in the field. Inuse, a test solution containing the appropriate analyte-liposomeconjugate, and known or suspected to contain the analyte, is spotted ordropped onto contact portion 402 of absorbent material 420. Contactportion 402 is wet with the test or control solution, or a carriersolution or wicking reagent after initial application of the test orcontrol solution, until the solution traverses the absorbent material420 from contact portion 402 to end-point indicator portion 408.Competitive binding between the analyte and the conjugate occurs incompetitive binding portion 402. Conjugate which is not bound incompetitive binding portion 422 is accumulated in measurement portion424 as a result of the binding reaction between the conjugate and thereceptor non-diffusively bound to measurement portion 424.

The signal from the marker may be visually read through window 416 overmeasurement portion 424. Alternatively, the signal from accumulatedliposome-encapsulated marker may be instrumentally read by, for example,a spectrophotometer which is adapted for use with the device inaccordance with the invention.

The present invention is applicable to procedures and products fordetermining a wide variety of analytes. As representative examples oftypes of analytes, there may be mentioned: environmental and foodcontaminants, including pesticides and toxic industrial chemicals;drugs, including therapeutic drugs and drugs of abuse; hormones,vitamins, proteins, including antibodies of all classes; peptides;steroids; bacteria; fungi; viruses; parasites; components or products ofbacteria, fungi, viruses, or parasites; allergens of all types; productsor components of normal or malignant cells; etc. As particular examples,there may be mentioned T₄ ; T₃ ; digoxin; hcG; insulin; theophylline;leutinizing hormones and organisms causing or associated with variousdisease states, such as streptococcus pyogenes (group A), Herpes SimplexI and II, cytomegalovirus, chlamydiae, etc.

As hereinabove indicated, the assay may be qualitative (presence orabsence of certain level of analyte) or quantitative orsemi-quantitative. The preparation of suitable standards and/or standardcurves (the term "standard curve" is used in a generic sense to includea color chart) is deemed to be within the scope of those skilled in theart from the teachings herein.

The method of the invention, and preparation and use of the test devicein accordance with the invention, are illustrated by the followingExamples.

EXAMPLES

Materials

Alachlor was purchased from Chem Service (West Chester, Pa.). Bovineserum albumin (BSA), N-succinimidyl-S-acetylthioacetate (SATA),dipalmitoyl phosphatidyl ethanolamine (DPPE), cholesterol,poly(vinylpyrrolidone) (PVP, 10,000 mol. wt.), Tween-20, triethylamine,Molybdenum Blue spray reagent, Isosulfan Blue and Sephadex G-50 werepurchased from Sigma (St. Louis, Mo.). Dipalmitoyl phosphatidyl choline(DPPC) and dipalmitoyl phosphatidyl glycerol (DDPG) were obtained fromAvanti Polar Lipids (Alabaster, Ala.). Sulforhodamine B was purchasedfrom Eastman (Rochester, N.Y.), and Fast Green FCF from Allied Chemical(New York, N.Y.). Carnation non-fat dry milk powder (CNDM) was obtainedlocally. Protein assay dye reagent, goat anti-rabbit IgG alkalinephosphatase conjugate and the substrates for alkaline phosphatase werepurchased from Bio-Rad (Hercules, Calif.). Egg white avidin was obtainedfrom Molecular Probes (Eugene, Oreg.). Whatman (Maidstone, UK) silicagel TLC flexible plates and preparatory silica get plates, bothcontaining fluorescent indicator, were used. Plastic-backednitrocellulose membranes with pore size >3 μm were obtained fromSchleicher and Schull (Keene, N.H.). A Paasch VL airbrush (Texas ArtSupply, Houston, Tex.) was used for applying the antibody and egg whiteavidin to the membrane. The rabbit anti-Alachlor IgG used in preliminaryinvestigations was supplied by ImmunoSystems (Scarborough, Me.).Subsequent supplies of antiserum were provided by the Cornell UniversityCollege of Veterinary Medicine.

EXAMPLE 1 Preparation of Materials and Reagents

Conjugate and antibody production.

Alachlor was conjugated to BSA by modification of a published method, asdescribed in Reeves, et al. Anal. Lett., 26 (1993) 1461, herebyincorporated by reference. Antibodies to the immunogenic conjugate wereraised in New Zealand white rabbits by standard procedures. Theantibodies produced were purified by the caprylic acid-ammonium sulfateprecipitation method, described in McKinney, et al., J. Immunol.Methods, 96 (1987) 271, hereby incorporated by reference.

Antibody and avidin immobilization.

In the strip assay, a protein-binding membrane (absorbent material) witha plastic backing to provide rigidity was required, and nitrocellulosemembrane supported in this manner was found to be the most suitable. Anairbrush was used to dispense the antibody and egg white avidinsolutions for immobilization. The membrane was cut to a desired size(7.9 cm high and a suitable width for later subdivision into strips 5 mmwide), thoroughly wetted with 10% methanolic TBS (tris buffered saline,pH 7.0) and dried before application of antibody and avidin solutions.The membrane sheet was mounted on a mobile platform that moved at aconstant rate in front of the airbrush used to spray the antibodysolution at a concentration between 0.2 and 1 mg ml⁻¹ (depending onpreparation) onto the competitive binding portion of the membrane, andegg white avidin solution at 1 mg ml⁻¹ onto the measurement portion ofthe membrane. The protein bands were allowed to vacuum dry for 1 hour.

After applying antibody and egg white avidin to the nitrocellulosesheets, it was necessary to block the membrane to reduce non-specificbinding and to aid the mobility of the liposomes. Both PVP and BSA werefound to be suitable for this purpose, but the former was less expensiveand more tractable and was routinely used as the blocking agent.Non-uniform migration of liposomes with certain batches of membrane, canbe alleviated by the use of very low levels of detergent in the blockingsolution. Tween-20 at 0.002% was found suitable for this purpose, and atthis level of application it did not cause lysis of the liposomes duringthe 8-min analysis run.

The coated nitrocellulose sheet was then immersed in blocking agent (asolution of 2% polyvinylpyrrolidone and 0.002% Tween-20 in TBS) for 1hour on a rotating shake and dried under vacuum for 3-4 hours. Preparedsheets were stored at 4° C. in the presence of silica gel desiccantuntil ready for use. The sheets were cut into strips using a papercutter when required. The final strips were 5×79 mm with a 5 mm longantibody zone 15 mm above the bottom of the strip and a similar eggwhite avidin zone 35 mm from the bottom.

Analyte-lipid conjugation.

To provide the requisite antigenic sites (epitopes) on the surface ofthe liposomes for the competitive assay format, it is necessary to forma conjugate between the analyte molecule and a lipid, DPPE, which isthen incorporated into the liposome bilayer.

For conjugation of the Alachlor to DPPE, a thiolating reagent, SATA,described in Duncan, et al., Anal. Biochem., 132 (1983) 68, which ishereby incorporated by reference, was used as the coupling agent basedon a modification of a procedure reported in Feng, et al., J. Agric.Food Chem. 38 (1990) 59, hereby incorporated by reference. Twenty mg ofDPPE were suspended in 3 ml of 0.7% triethylamine in chloroform andsonicated under nitrogen for 1 minute in a 45° C. bath. To the DPPE, 2molar equivalents of SATA in 1 ml of the same solvent were added slowly.The reaction flask was capped and stirred at room temperature for ca. 20minutes, the end point of the reaction being indicated by the clearingof the mixture. The solvent was removed on a rotary vacuum evaporator,and 2 ml of 30 mM hydroxylamine hydrochloride in methanol, adjusted topH 8.2 with NaOH, were added. The reaction mixture was vortexedvigorously and stirred at 45° C. for 1 hour under nitrogen, maintainingthe pH at 8.2 using dilute NaOH in methanol. A 2.8 molar excess (toDPPE) of Alachlor in 1 ml of 30 mM hydroxylamine, pH 8.2 in methanol,was added to the reaction flask. The reaction mixture was stirred at 45°C. for 2 hours, with the pH being maintained at 8.2, and the reactionwas allowed to continue at 45° C. overnight (ca. 17 hours). The productwas purified on a preparatory silica gel plate using the solvent systemchloroformacetone-methanol-glacial acetic acid-water (60:20:20:5:4,v/v).The purified Alachlor-DPPE conjugate was quantified by Bartlett'sphosphorous assay described in Bartlett, J. Biol. Chem., 234 (1959) 466,which is hereby incorporated by reference.

Because radioactive Alachlor was not available for use as a tracer,confirmation of the successful conjugation reaction was achieved by acombined thin-layer chromatographic and an enzyme immunostaining methoddescribed in Mattsby-Baltzer, Eur. J. Biochem., 138 (1984) 333, herebyincorporated by reference, using an anti-Alachlor antibody supplied byImmunoSystems. The procedure involved the duplicate chromatographicanalysis of the reaction mixture on TLC plates. Whatman Silica Gel/UVplates were prewashed in the solvent described above and dried, and thesamples were run in the same solvent. One of the TLC plates was dried,blocked for 1 hour in a solution of 1% BSA and 0.5% CNDM in TBS, washedthree times for 10 minutes each in TBST (TBS containing 0.05% Tween-20),and placed overnight in a solution containing the antibody to Alachlor(20 μg/ml in TBST). The plate was washed three times for 10 min each inTBST and placed in a solution containing a goat anti-rabbit alkalinephosphatase conjugate (stock diluted 1:3000 with TBST containing 0.02%BSA) for 2 hours. The plate was washed as before and developed with thesubstrate for alkaline phosphatase (nitroblue tetrazolium in aqueous DMFwith magnesium chloride and 5-bromo-4-chloro-3-indolyl phosphate in DMF,prepared according to the manufacturer's instructions). When colordevelopment was complete (10 min), the plate was washed in distilledwater and dried. A purple spot indicated the presence of Alachlor. Theother TLC plate was sprayed with molybdenum blue reagent (1.3%molybdenum oxide in 4.2M sulfuric acid) which is specific forphospholipids. The Alachlor-DPPE spot appeared purple with the alkalinephosphatase substrate stain and blue with the molybdenum blue sprayreagent.

Preparation of dye-encapsulated Alachlor-tagged liposomes.

Liposomes were formed by the reversed-phase evaporation method, asdescribed in Szoka, et al., Biochim. Biophys. Acta, 601 (1980) 559, andO'Connell, et al., Anal. Chem., 31 (1985) 142, the disclosures of whichare hereby incorporated by reference, from a mixture of DPPC,cholesterol, DPPG, and Alachlor-DPPE conjugate in a molar ration of5:5:0.5:0.01. Forty-three μmol of this mixture were dissolved in 4.2 mlof a solvent mixture containing chloroform-isopropyl ether-methanol(6:6:1,v/v). This solution was warmed to 45° C. and 0.7 ml of the dyesolution was added with swirling. This mixture was sonicated for 5minutes under a low flow of nitrogen. The organic phase was removedunder vacuum on a rotary evaporator at 40° until all frothing hadstopped. An additional 1.3 ml aliquot of the dye solution was added, andthe liposomes were then sequentially extruded twice through each of twopolycarbonate filters of decreasing pore sizes of 1.0 μm and 0.4 μm. Thediameters of the liposome preparations were measured by laser scatteringin a LA-900 particle size distribution analyzer (Horiba, Irvine,Calif.), using the manufacturers method, except that the usualsonication step was omitted to avoid lysis (rupture) of the liposomes.Finally, to remove any unencapsulated dye, the liposomes were gelfiltered on a 1×14 cm Sephadex G-50 column and dialyzed overnightagainst TBS at 4° C. When stored at 4° C., there was no significantleakage of dye over a period of 9 months as described below.

Sulforhodamine B was chosen as the dye for encapsulation, as describedin O'Connell, above, and Chen, et al., Anal. Biochem., 172 (1988) 61,hereby incorporated by reference, because of its fluorescence and highvisible extinction coefficient. To prepare the dye, 20 mM Tris was usedto buffer the dye solution. The pH was adjusted to 7.0 with NaOH toeffect dissolution. The final solution contained 100 mM dye in 20 mMTris at a pH of 7.0 with an osmolarity approximately equal to TBS, whichwas the buffer routinely used in all aqueous operations of theexperiments. In some experiments 200 mM Sulforhodamine B was used togive a greater color intensity on the strips.

Because Sulforhodamine B is highly fluorescent and this fluorescenceundergoes self-quenching when encapsulated, the integrity of theliposomes can be determined by measuring fluorescence intensity beforeand after lysis. Total and almost instantaneous lysis of the liposomeswas effected by addition of a solution (final concentration=30 mM) ofn-octyl-β-D-glucopyranoside at room temperature. For these fluorescenceexperiments, the dye was excited at a wavelength of 543 nm andfluorescence measured at the emission wavelength of 596 nm.

In some experiments the non-fluorescent dyes Isosulfan Blue and FastGreen FCF were encapsulated by the same methods as Sulforhodamine B.

Characteristics of liposomes

Liposomes were prepared by the reversed-phase evaporation methoddescribed in Szoka and O'Connell, above, but without extrusion throughpolycarbonate filters, giving a high yield of liposomes. However, theseheterogeneously sized liposomes did not migrate evenly on the teststrips used in the assay. This was improved by passing the preparationssequentially twice through each of two polycarbonate filters of 1.0 and0.4 μm nominal pore diameter. Liposomes passed only through the 1.0 μmfilter had a mean diameter of 1.82 μm, with a standard deviation of 0.8,while those passed through both filters had a mean diameter of 0.68 μmwith a standard deviation of 0.12. This discrepancy between the size ofthe pores on the polycarbonate filters and the final size of theliposomes is not surprising, as the liposomes are very flexible, and canthus "squeeze" through a pore of smaller diameter. The liposomes of 0.68μm diameter were a much more homogeneous population than those of 1.82μm diameter, and both populations migrated more evenly on thenitrocellulose sheets than did the unextruded liposomes. Passing thepreparation through an even smaller sized filter (0.2 μm) did notimprove the migration behavior, and reduced the yield considerably.Consequently, liposomes that had been passed through the 1.0 and 0.4 μmfilters were used in all subsequent experiments.

The absorption spectrum of dilute, free Sulforhodamine B gave a peak at566 nm, with a shoulder at 532 nm. The intact liposomes, containing dyeat a concentration sufficiently high to form dimers, as described inChen, et al., Anal. Biochem., 172 (1988) 61, hereby incorporated byreference, gave a spectrum with peaks at 532 and 568 nm, with the 568 nmpeak at 70% of the height of the 532 nm peak. The addition of surfactantto the liposomes caused lysis of the liposomes and consequent dilutionof the dye, and thus converted the spectrum to that of the free dye.

The presence of Alachlor on the surface of the liposomes wasdemonstrated by the reversal, by free Alachlor, of antibody-inducedaggregation of the liposomes. The aggregated liposomes could beprecipitated by centrifugation.

The liposomes were stored at 4° C., and the temporal stability wasstudied over time by measuring the percentage of free dye in thepreparation, thereby allowing calculation of the percentage of theliposomes that had lysed.

The characteristics of the liposomes used in these studies are shown inTable 1. From the size measurement results, it is possible to calculatethat the average volume of a single liposome is 1.7×10⁻¹⁰ μl. Byassuming the dye encapsulated was equal in concentration to the originaldye solution used, and by comparing the fluorescence of lysed liposomesto that of standard Sulforhodamine B solutions, it is possible tocalculate that there were ca. 1.2×10⁸ liposomes μl⁻¹ and that eachliposome contained ca. 9.6×10⁶ molecules of dye. Assuming that theaverage surface area of the DPPC molecules is 71 Å², and that ofcholesterol molecules in a mixed bilayer is 19 Å², as described inIsaraelachvili, eta al., Biochim. Biophys. Aeta, 389 (1975) 13, andgiven that the DPPE-Alachlor is 0.1 mole % of the total lip, there areca. 3500 molecules of Alachlor on the outer surface of a singleliposome.

                  TABLE 1                                                         ______________________________________                                        Liposome Characteristics                                                      ______________________________________                                        Mean diameter (± S.D.)                                                                         0.68 ± 0.12 μm                                      Volume              1.7 × 10.sup.-10 μl                              Liposome conc.      1.2 × 10.sup.8 lipo μl.sup.-1                    SRB.sup.a conc.     100 mM                                                    SRB.sup.a (molecular).sup.b                                                                       9.6 × 10.sup.6 molec. lipo.sup.-1                   Alachlor conc..sup.c                                                                              3.5 × 10.sup.3 molec. lipo.sup.-1                   Stability           >9 months                                                 ______________________________________                                         .sup.a Sulforhodamine B.                                                      .sup.b The number of molecules of SRB per liposome.                           .sup.c The number of molecules of Alachlor on the outer surface of a          single liposome containing 0.1 mole % DPPEAlachlor.                      

EXAMPLE 2 Assay format

The assay device configuration consists of a wicking reagent containingAlachlor-tagged liposomes and a test strip comprised of a wick, animmobilized anti-Alachlor zone and an egg white avidin capture zone insequence. The assay is performed by dispensing 100 μl (2 drops) of thesample or control solution and 50 μl (1 drop) of a three timesconcentrated TBS buffer into a 10×75 mm glass test tube, mixing thecontents, and adding 50 μl (1 drop) of a liposome solution (stockliposome solution diluted 1:50, dilution varying according topreparation). The test tube is shaken mildly to mix the contents and thetest strip is inserted into the tube; the strip is left in the tubeuntil the solution front reaches the end of the strip (about 8 min); thestrip is removed and air dried. The color intensity of the antibody zoneand the egg white avidin zone are estimated either visually or byscanning densitometry, as described in Reeves, (1993).

The measurement of the extent of the competitive binding reactions ofthe analyte molecules and the tagged liposomes to the immobilizedantibodies was optical. Visual estimation of the color intensity can beused, but for more accurate quantitation during development it was foundto be preferable to use a computer scanner and Scan Analysisdensitometry software (Biosoft, Ferguson, Mo.) to convert the redcoloration into greyscale readings that can be measured.

A series of analyte determinations were made as described above with aseries of Alachlor standards of varying concentrations. A decrease incolor of the antibody zone with increasing concentrations of addedAlachlor, and a concomitant increase in the color of the egg whiteavidin zone, was observed.

Dose-response data obtained by scanning densitometry of strips run inthe presence of various concentrations of Alachlor are shown in FIG. 5,which is a graph of greyscale density versus Alachlor concentration(ppb), measured in both the antibody and avidin zones. The response inboth the antibody and avidin zones varied logarithmically when measuredusing scanning densitometry, and both were estimated to be able todetect 5-10 μg/1 Alachlor. When these strips were assessed visually, asimilar determination could be made, but at low levels of added Alachlorit was somewhat easier to detect increases of red color over a whitecontrol (avidin zone) than decreases in color intensity (antibody zone).

These examples are included to illustrate the practice of the invention.However, numerous variations and alternatives for the structure,components and use of the test device of the invention are possible. Forexample, a test device packaged as part of a commercial kit mightinclude special holders for individual strips, in which openings areprovided for sample application and optical readout, for example, asshown in FIG. 4. In such a configuration, the strips could be run in anyorientation, e.g., lateral, instead of vertical flow.

In an alternative embodiment of the invention, a dual-strip test deviceas shown in FIG. 2 may be employed. In this design, after application ofthe protein zones, the strip is divided in two by removal of a very thinstrip of nitrocellulose from the plastic backing, thereby providing twoidentical strips with a hydrophobic separation to prevent solutioncross-talk. A tolerance level control is applied to the strip adjacentto the sample, and both are run simultaneously. This verifies the stripperformance and provides a more quantitative interpretation of the stripresults. As described above, multi-analyte assays may also be conductedusing the test device and method of the invention.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed is:
 1. A method for detecting or determining an analyte,comprising:providing a test device comprising an absorbent material,which absorbent material comprises:a contact portion proximate to afirst end of said absorbent material; a measurement portion at alocation on said absorbent material which is positioned away from thefirst end, said measurement portion having a receptor non-diffusivelybound to said measurement portion, wherein said receptor is capable ofbinding to a conjugate of liposomes and the analyte, wherein a pluralityof molecules of the analyte is attached to a single liposome, andwherein said liposomes comprise a detectable marker in the interior ofsaid liposomes; a competitive binding portion positioned between andsegregated from said contact and said measurement portions on saidabsorbent material and having a binding material for the analytenon-diffusively bound to said competitive binding portion; and aterminal portion positioned adjacent to said measurement portion andfarther from the first end than said measurement portion, wherebymaterial which is not immobilized in said measurement portion passesinto said terminal portion, wherein said contact, measurement,competitive binding, and terminal portions are located on a singleabsorbent material; contacting a solution of the analyte and theconjugate with said contact portion of said absorbent material; allowingthe solution to migrate from said contact portion into said terminalportion of said absorbent material; and determining the amount of saidmarker in said measurement portion of said absorbent material as ameasure of said analyte in a sample.
 2. A method according to claim 1,wherein said contacting is carried out by inserting the contact portionof said absorbent material into said test solution.
 3. A methodaccording to claim 1, wherein said contact portion further comprises awicking portion at the first end, said contacting is carried out byspotting said test solution onto said absorbent material in said contactportion outside of said wicking portion, and said allowing comprisesinserting said wicking portion into a wicking reagent.
 4. A methodaccording to claim 1, wherein said determining is carried out bymeasuring an absolute concentration of said analyte in said sample.
 5. Amethod according to claim 1, wherein said determining is carried out bycomparing the amount of said marker in said measurement portion with areference standard having a known concentration of said analyte toevaluate the concentration of said analyte in said sample relative tosaid known concentration.
 6. A method according to claim 1, wherein saidmarker is a fluorescent dye, a visible dye, a bioluminescent material, achemiluminescent material, a radioactive material, or an enzymaticsubstrate.
 7. A method according to claim 6, wherein said marker is afluorescent dye or a visible dye and said determining is carried out byfluorimetric or spectrophotometric measurement.
 8. A method according toclaim 7, wherein said marker is a visible dye and said determining iscarried out visually with the unaided eye.
 9. A method according toclaim 1, wherein said liposomes are prepared from one or morephospholipids, glycolipids, steroids, alkyl phosphates, or fatty acidesters.
 10. A method according to claim 9, wherein said phospholipidsare selected from the group consisting of lecithin, sphingomyelin, anddipalmitoyl, and said steroids are selected from the group consisting ofcholesterol, chlorestanol, and lanosterol.
 11. A method according toclaim 1, wherein said liposomes are prepared from a mixture comprisingdipalmitoylphosphatidylcholine, cholesterol, anddipalmitoylphosphatidylglycerol.
 12. A method according to claim 1,wherein said analyte is an antigen or hapten, and said binding materialis an antibody for said antigen or said hapten.
 13. A method accordingto claim 1, wherein said analyte is alachlor, PCB's, dioxin, a hormone,a vitamin, a metabolite, or a pharmacological agent.
 14. A methodaccording to claim 1, wherein said determining comprises lysing saidliposomes to release said marker.